Project Summary/Abstract Voltage-gated proton (Hv) channels carry robust proton currents across membranes and are gated by both voltage and transmembrane pH gradient (?pH). They normally serve as acid (proton) extruder and play a critical role in pH homeostasis of metabolically active cells. For example, in phagocytes, Hv channels compensate charge and pH imbalance during respiratory burst of NADPH oxidase to promote the production of reactive oxygen species (ROS) for pathogen defense. In human sperms, human Hv1 (hHv1) channel triggers intracellular alkylation essential for sperm motility and capacitation. hHv1 channel also highly correlates with cancer invasiveness and ischemic neuronal cell death, for their association with NADPH oxidase in these cells. Voltage and ?pH gating are two fundamental biophysical properties determining the dynamics of proton currents through Hv channels, which in turn underlie their physiological and pathophysiological roles in the cells mentioned above. Hv channels are standalone voltage sensors and adopt structures similar to these from voltage-gated cation channels. The ion selectivity, voltage and ?pH gating of Hv channels have been characterized by patch clamp electrophysiology in detail with many key residues been identified. However, the conformational changes and dynamics induced by voltage and pH to gate channel pore remain unknown, which becomes a critical barrier to understand the mechanisms of Hv channel gating. In the present project, by working on the purified hHv1 proteins in liposomes, we are able to impose and switch electrical voltage and pH gradients on hHv1 channels and then examine their conformational transitions and dynamics by single- molecule FRET (smFRET). Furthermore, we will use liposome fluorescence flux assay to examine the function of hHv1 channels under the conditions identical to these for smFRET studies. These studies will reveal the conformational changes of hHv1 channels induced by voltage and ?pH and their direct relevance with channel pore gating. More importantly, smFRET studies can reveal the structural dynamics, i.e the scheme and rates of the hHv1 conformational transitions, which are unattainable from static conformations provided by X-ray crystallography and cryo-EM. In addition, we will also investigate the structural basis of hHv1 inhibition by small molecule inhibitors, such as Zn2+ and guanidine derivative 2-guanidinobenzimidazole (2GBI), using the same smFRET approach. These studies will help us to understand the structural basis underlying hHv1 channel gating and inhibition, and to provide novel mechanistic insights to predict the function of hHv1 channel under different physiological and pathophysiological conditions in phagocytes, sperms, microglia, and cancer cells. Since voltage sensors of all voltage-gated cation channels, including the hHv1 channel, are highly conserved. Therefore, my project will also make conceptual and methodological frameworks to understand voltage sensing and gating in voltage-gated K, Na and Ca channels.